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S Citi

Publications and source records attributed to S Citi.

At least 19 recordsLinked to original sources
PubMed1992

Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells.

When epithelial cell cultures are transferred from a medium with a normal extracellular calcium concentration (1-2 mM) to a medium with a low extracellular calcium concentration (LC, less than 50 microM free Ca2+) cell-cell contacts are disrupted, and the tight junction-dependent transepithelial resistance drops. In this study, I used MDCK epithelial cells to investigate the effects of LC on the localization of the tight junction protein cingulin, and the role of protein kinases in the events induced by LC. Immunofluorescence analysis showed that within 15 min of incubation of confluent monolayers in LC, cingulin labeling was dislocated from the cell periphery, as an array of granules forming a ring-like structure. At later times after calcium removal, cingulin labeling appeared mostly cytoplasmic, in a diffuse and granular pattern, and cells appeared rounded and smaller. These events were not influenced by lack of serum, or by preincubation with 10 mM sodium azide or 6 mg/ml of cycloheximide. However, the disruption of cell-cell contacts, the cell shape changes, and the redistribution of cingulin and other junctional proteins induced by LC were inhibited when cells were pretreated with the protein kinase inhibitor H-7 (greater than or equal to 30 microM). The inhibitors H-8 and, to a lesser degree, staurosporine were also effective, whereas HA-1004 and ML-7 showed essentially no activity, suggesting a specificity of action of different inhibitors. Measurement of the transepithelial resistance showed that the kinase inhibitors that could prevent junction disassembly could also reduce the drop in transepithelial resistance induced by LC. Dose-response curves demonstrated that H-7 is the most effective among the inhibitors, and the transepithelial resistance was 70% of control up to 1 h after calcium removal. These results suggest that low extracellular calcium modulates junctional integrity and cytoskeletal organization through an effector system involving protein kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Polarized functions and permeability properties of rat epididymal epithelial cells in vitro.

Cultured rat caput and cauda epididymidal epithelial cells are shown to exhibit polarized properties characteristic of functioning epithelia. When grown on plastic substrates coated with reconstituted basement membrane, confluent monolayers of cells from both regions formed domes characteristic of other transporting epithelia. Immunocytochemical localization of three proteins characteristically associated with epithelial junctional complexes revealed that uvomorulin, zonula occludens 1 and cingulin were present in cultured epididymal epithelial cells and that their distribution was similar to that in the epididymal epithelium in vivo. These three molecules were not found in epididymal stromal cells. Cells from both regions growing in two compartment chambers developed an electrical resistance across the monolayer with a magnitude characteristic of high resistance epithelia. The optimal plating density of cells was 0.75 x 10(6) cells cm-2. The presence of reconstituted basement membrane on the filters did not affect the resistance of the cells. Inulin passage from basal to apical chambers was less than 2% over 24 h. These results show that several polarized functions of epididymal epithelial cells can be maintained in culture and that this type of culture system is useful for studying the function of the epididymis in vitro.

Animals↗
PubMed1991

The role of phosphorylation in development of tight junctions in cultured renal epithelial (MDCK) cells.

We have explored the effect of the protein kinase inhibitor H7 on tight junction formation in a MDCK cell model for the development of cell-cell contact, tight junctions and epithelial polarity: the "Ca++ switch" model. In this developmental model, which is thought to mimic processes during the early morphogenesis of epithelial tissues, the protein kinase inhibitor H7 markedly inhibits the development of transepithelial resistance of confluent MDCK cells during the "switch" from low (1-5 microM) to normal (1.8 mM) Ca++ media compared with control MDCK cells. Moreover, indirect immunofluorescence using specific antisera against two tight junctional proteins, ZO1 and cingulin, revealed that H7 inhibits the sorting of these proteins from an intracellular site to the lateral surfaces of MDCK cells when the Ca++ in the medium is raised. These data suggest protein kinase mediation in sorting events that lead to the assembly of tight junctions.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Localization of myosin in the cytoskeleton of brush border cells using monoclonal antibodies and confocal laser-beam scanning microscopy.

Monoclonal antibodies binding to the rod portion of brush border myosin were used to localize myosin in chicken intestinal brush border cells by indirect immunofluorescence. Isolated cells, or cells still attached in a sheet, were analyzed by conventional epifluorescence microscopy, which showed that most of the immunoreactive myosin is localized in the apical brush border (terminal web), and in a basal region. In addition, a weak, diffuse granular and rod-like labeling was detected throughout the cell body. Using the laser-scanning confocal microscope (White et al., 1987), a more precise localization of the myosin within the terminal web and the cell body was obtained. In the terminal web, most of the myosin was concentrated in a circumferential ring, below the plasma membrane, and the remaining myosin was found in the inter-rootlet area. These two populations of myosin were topologically strictly related, since they were found in the same optical sections. In the cell body, as well as in the basal region, the myosin was found to be associated with the outer limiting membrane of the cell, in a cortical location, whereas essentially no myosin was detected in the cytoplasm.

Animals↗
PubMed1991

Cingulin, a specific protein component of tight junctions, is expressed in normal and neoplastic human epithelial tissues.

Cingulin is a 140-kd protein localized on the cytoplasmic face of avian tight junctions. The expression of cingulin in human normal and neoplastic colonic tissue has been investigated with an antiserum against chicken cingulin. Human cingulin shares its apparent molecular mass and localization with avian cingulin. In normal colonic epithelium, villous adenomas, and differentiated adenocarcinomas, cingulin staining is observed in the junctional region of the polarized cells lining the surface, the crypts, and the glandular lumina. In poorly differentiated adenocarcinomas, labeling also is observed at the interface between cancer tissue and stroma, or in clumps of malignant cells, forming a pattern that highlights the presence of small, compressed lumina. The cingulin content of four adenocarcinomas, estimated by immunoblotting and densitometry, was higher than that of the normal tissue (150% to 230%). Cingulin was detected in a metastasis from a colon adenocarcinoma but not in nonepithelial tissues and neoplasias, suggesting that cingulin may be a useful marker in the characterization of colonic and probably other epithelial neoplasias.

Adenocarcinoma↗
PubMed1989

Parallel modulation of brush border myosin conformation and enzyme activity induced by monoclonal antibodies.

Monoclonal antibodies binding to distinct epitopes on the tail of brush border myosin were used to modulate the conformation and state of assembly of this myosin. BM1 binds 1:3 of the distance from the tip of the tail to the head and prevents the extended-tail (6S) monomer from folding into the assembly-incompetent folded-tail (10S) state, whereas BM4 binds to the tip of the myosin tail, and induces the myosin to fold into the 10S state. Thus, at physiological ionic strength BM1 promotes and BM4 blocks the assembly of the myosin into filaments. Using BM1 and BM4 together, we were able to prevent both folding and filament assembly, thus locking myosin molecules in the extended-tail 6S monomer conformation at low ionic strength where they normally assemble into filaments. Using these myosin-antibody complexes, we were able to investigate independently the effects of folding of the myosin tail and assembly into filaments on the myosin MgATPase. The enzymatic activities were measured from the fluorescent profiles during the turnover of the ATP analogue formycin triphosphate (FTP). Extended-tail (6S) myosin molecules had an FTPase activity of 1-5 X 10(-3) s-1, either at high ionic strength as a monomer alone or when complexed with antibody, or at low ionic strength as filaments or when maintained as extended-tail monomers by the binding of BM1 and BM4. Folding of the molecules into the 10S state reduced this rate by an order of magnitude, effectively trapping the products of FTP hydrolysis in the active sites.

Animals↗
PubMed1989

ZO-1 and cingulin: tight junction proteins with distinct identities and localizations.

The relative localization of ZO-1 and cingulin, the only two known components of the tight junction, was compared in Madin-Darby canine kidney (MDCK) cells, chicken small intestine, rat kidney distal convoluted tubule, and a hepatoma cell line. Immunoblot analysis demonstrated that cingulin and ZO-1 are immunologically unrelated and that, in the colon, cingulin is a single polypeptide with a molecular mass of 140 kDa. Immunofluorescent localization of cingulin and ZO-1 in confluent monolayers of MDCK cells showed identical staining patterns. However, subconfluent MDCK cells showed distinct localizations of the two proteins. Both cingulin and ZO-1 were found at the plasma membrane only at areas of cell-cell contact, but cingulin was diffusely distributed within the cytoplasm, whereas ZO-1 showed a more clustered internal arrangement. Cingulin and ZO-1 were identically localized at the plasma membrane of hepatoma tissue culture (HTC) cells at sites of cell-cell contact. In chicken intestine examined at the ultrastructural level, immunogold particles associated with cingulin were found approximately three times farther from the junctional membrane than those affiliated with ZO-1.

Animals↗
PubMed1989

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.

Animals↗
PubMed1988

Active site trapping of nucleotide by smooth and non-muscle myosins.

The folded 10 S monomer conformation of smooth muscle myosin traps the hydrolysis products ADP and Pi in its active sites. To test the significance of this, we have searched for equivalent trapping in other conformational and assembly states of avian gizzard and brush border myosins, using formycin triphosphate (FTP) as an ATP analogue. When myosin monomers were in the straight-tail 6 S conformation, the hydrolysis products were released at about 0.03 s-1. Adoption of the folded 10 S monomer conformation reduced this rate by more than 100-fold, effectively trapping the products FDP and Pi in the active sites. This profound inhibition of product release occurred only on formation of the looped tail monomer conformation. In vitro-assembled myosin filaments released products at a comparable rate to free straight-tail 6 S monomers, and smooth muscle heavy meromyosin, which lacks the C-terminal two-thirds of the myosin tail, also did not trap the products in this way. Phosphorylation of the myosin regulatory light chain had no effect on the rate of product release from straight-tail 6 S myosin monomers or from myosin filaments. Rather, it allowed actin to accelerate product release. Phosphorylation acted also to destabilize the folded monomer conformation, causing the recruitment of molecules from the pool of folded monomers into the myosin filaments. The two processes of contraction and filament assembly are thus both controlled in vitro by light-chain phosphorylation. A similar linked control in vivo would allow the organization of myosin in the cell to adapt itself continuously to the pattern of contractile activity.

Adenosine Triphosphate↗
PubMed1988

Cingulin, a new peripheral component of tight junctions.

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.

Amino Acids↗
PubMed1988

Brush border myosin filament assembly and interaction with actin investigated with monoclonal antibodies.

Monoclonal antibodies binding to epitopes in the rod portion of brush border myosin were used to study the mechanism of filament assembly and its role in myosin interaction with actin. The antibodies and their Fab fragments had specific effects on the size of the filaments assembled in vitro. Two antibodies (BM3 and BM4), directed against the tip of the myosin tail, completely inhibited myosin filament assembly. The other antibodies (BM1, BM2 and BM5), binding to other sites along the myosin rod, only partially blocked filament growth, and short filaments could be assembled. Thiophosphorylated brush border myosin filaments appeared slightly more stable to the effects of the antibodies than those composed of dephosphorylated myosin. Only one (BM3) of the antibodies which completely inhibited the assembly of new filaments was capable of disassembling preformed myosin filaments. The other antibody, BM4, partially disassembled filaments, leaving approximately 0.2-microns long 'cores', suggesting that polymerization in this myosin occurs by a biphasic mechanism, i.e. the formation of a stable nucleus of antiparallely packed molecules, followed by elongation. The antibodies BM1 and BM2 bound to myosin filaments generating a regular transverse pattern with a approximately 14-nm periodicity, and had little effect on the stability of these preformed filaments. Inhibition of filament formation and solubilization of the myosin by the antibodies appeared to be associated with inhibition of myosin interaction with actin, as measured by the actin-activated MgATPase activity. In the presence of the antibodies which completely inhibit filament assembly, we observed a decrease to approximately 20% (BM4-Fab) and to approximately 50% (BM3) of the control actin-activated myosin MgATPase activity, and this activity was kinetically different from that of the soluble myosin S1 fragment, suggesting that the rod has a profound effect on the kinetics of actomyosin interaction.

Actins↗
PubMed1988

Modulation of cellular morphology and locomotory activity by antibodies against myosin.

Three monoclonal antibodies directed against chicken brush border myosin were used to study the possible function of myosin in microfilament organization and locomotion of chicken fibroblasts. These antibodies bind to distinct and separate epitopes on the heavy chain of chicken nonmuscle myosin and display differential effects of myosin filament formation and actin-myosin interaction (Citi, S., and J. Kendrick-Jones. 1988. J. Musc. Res. Cell Motil. 9: 306-319). When injected into chicken fibroblasts, all antibodies induced breakdown of stress fibers. Concomitantly, a large proportion of the cells developed extensive lamellae which altered their morphology drastically. These cells showed also increased locomotory activity. All effects were concentration dependent and reversible. The most drastic alterations were observed with cells injected with antibody quantities exceeding the quantity of cellular myosin (molar ratios of antibody to myosin greater than 3:1). The finding that antibodies with different effects on myosin filament formation in vitro all induce similar intracellular processes suggests that it is the antibody-induced decrease in functional myosin that triggers an increase in plasma membrane dynamics and locomotory activity, rather than differences in myosin filament length or conformation.

Actin Cytoskeleton↗
PubMed1987

Polymerization of vertebrate non-muscle and smooth muscle myosins.

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers (Cm) and filaments (Cp) present under a given set of conditions as a function of the total myosin concentration (Ct). Below 200 mM-NaCl, each myosin displayed distinct "critical monomer concentrations" (Cc) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 mM-NaCl, MgATP increased the Cc of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their Cc values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer in equilibrium 6 S monomer in equilibrium 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.

Animals↗
PubMed1987

Effects of light chain phosphorylation and skeletal myosin on the stability of non-muscle myosin filaments.

The effect of light chain phosphorylation and the presence of skeletal muscle myosin on the stability of non-phosphorylated non-muscle myosin filaments was investigated. Purified skeletal, brush border and thymus myosins were assembled in vitro into hybrid filaments consisting of varying proportions of (1) non-muscle and skeletal myosins, or (2) phosphorylated and non-phosphorylated non-muscle myosins. The stability of these hetero- and homopolymers in the presence of MgATP was determined using sedimentation, gel electrophoresis and immunochemical techniques. In addition, the effect of a monoclonal antibody, binding to the tip of brush border myosin tail, on the assembly of the homo- and heteropolymers, was tested. Filamentous non-phosphorylated non-muscle myosin was disassembled by MgATP to the same extent whether in homo- or heteropolymers, indicating that skeletal myosin has no stabilising effect on the hybrid filaments. The presence of small amounts of phosphorylated non-muscle myosin was, however, found to prevent the complete disassembly by MgATP of non-phosphorylated non-muscle myosin filaments, indicating that light chain phosphorylation stabilizes co-operatively non-muscle myosin filaments. The monoclonal antibody prevented the assembly of brush border myosin into both homo- and heteropolymers, and its effect on the filaments was compared with that of MgATP.

Adenosine Triphosphate↗
PubMed1987

Studies on the structure and conformation of brush border myosin using monoclonal antibodies.

We have produced and characterised five monoclonal antibodies against myosin isolated from chicken intestinal epithelial brush border cells. The binding sites of the antibodies on the rod portion of brush border myosin were localised using rotary shadowing/electron microscopy of myosin-antibody complexes. Two antibodies were shown to bind to the tip of the myosin tail, two antibodies to sites about two thirds down the length of the rod, and one antibody about one third down the length of the rod. Brush border myosin was digested with papain, trypsin and alpha-chymotrypsin, and they myosin fragments obtained were analysed by western blots with the monoclonal antibodies and polyclonal antiserum, and by gel overlay with 125I-labelled light chains. Using this approach we were able to identify and map the protease cleavage sites and thus characterise the proteolytic substructure of brush border myosin. Solid-phase assays, western blots and immunofluorescence were used to study the cross-reactivity of these monoclonal antibodies against a variety of myosins from different species and cell types, to assess the immunological relatedness between brush border myosin and homologous molecules present in different tissues and species. Finally, we used a competitive solid-phase assay to measure the 'relative affinities' of the antibodies towards the three possible conformational states of brush border myosin, i.e. filament, extended monomer and folded monomer.

Animals↗
PubMed1986

Regulation in vitro of brush border myosin by light chain phosphorylation.

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.

Animals↗